RESUMO
Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.
Assuntos
Replicação do DNA , DNA de Cadeia Simples , Humanos , DNA de Cadeia Simples/genética , Replicação do DNA/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Ligação Proteica , Ubiquitinação , Dano ao DNA , Instabilidade Genômica , DNA Helicases/genética , DNA Helicases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.
RESUMO
Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.
Assuntos
Ácidos Nucleicos Heteroduplexes/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Polarização de Fluorescência , Mutação , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Domínios Proteicos , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/químicaRESUMO
Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Lipoxigenase , Oxilipinas , Arabidopsis/metabolismo , Ácido Araquidônico , Ácidos Graxos Insaturados , Ácido Linoleico , Lipoxigenase/química , Lipoxigenase/genética , Lipoxigenase/metabolismo , Mutação , Oxilipinas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO-SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.
Assuntos
Corpos Nucleares , Proteína da Leucemia Promielocítica , Proteína SUMO-1 , Zinco , Motivos de Aminoácidos , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Zinco/químicaRESUMO
Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7's membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other's lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot-Marie-Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas rab de Ligação ao GTP , Doença de Charcot-Marie-Tooth/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Ubiquitin-like proteins (Ubl's) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.
Assuntos
Ubiquitina/metabolismo , Domínio Catalítico , Humanos , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Especificidade por Substrato , Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.
Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA , DNA de Cadeia Simples/genética , Recombinação Homóloga , Proteínas Nucleares/genética , Fatores de Processamento de RNA/genética , Proteína de Replicação A/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Proteína de Replicação A/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The ligase Itch plays major roles in signaling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding are critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPXY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pulldown assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with endophilins, amphyphisins, and pacsins but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modeling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with ß-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR toward its most common SH3 domain-containing partners and demonstrate that the PRR region is sufficient for binding.
Assuntos
Modelos Moleculares , Proteínas Repressoras/química , Ubiquitina-Proteína Ligases/química , Domínios de Homologia de src , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.
Assuntos
Ácido Aspártico/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/química , Liases/química , Modelos Moleculares , Proteínas Mutantes/química , Compostos Organomercúricos/metabolismo , Substituição de Aminoácidos , Ácido Aspártico/química , Bacillus megaterium/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cobre/química , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Liases/genética , Liases/metabolismo , Mercúrio/química , Mercúrio/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Compostos Organomercúricos/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismoRESUMO
A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.
Assuntos
Técnicas de Química Sintética/métodos , Escherichia coli/metabolismo , Biossíntese Peptídica/fisiologia , Peptídeos/metabolismo , Motivos de Aminoácidos , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Estrutura Terciária de Proteína , Tiorredoxinas/metabolismoRESUMO
XPC/Rad4 (human/yeast) recruits transcription faction IIH (TFIIH) to the nucleotide excision repair (NER) complex through interactions with its p62/Tfb1 and XPB/Ssl2 subunits. TFIIH then recruits XPG/Rad2 through interactions with similar subunits and the two repair factors appear to be mutually exclusive within the NER complex. Here, we show that Rad4 binds the PH domain of the Tfb1 (Tfb1PH) with high affinity. Structural characterization of a Rad4-Tfb1PH complex demonstrates that the Rad4-binding interface is formed using a motif similar to one used by Rad2 to bind Tfb1PH. In vivo studies in yeast demonstrate that the N-terminal Tfb1-binding motif and C-terminal TFIIH-binding motif of Rad4 are both crucial for survival following exposure to UV irradiation. Together, these results support the hypothesis that XPG/Rad2 displaces XPC/Rad4 from the repair complex in part through interactions with the Tfb1/p62 subunit of TFIIH. The Rad4-Tfb1PH structure also provides detailed information regarding, not only the interplay of TFIIH recruitment to the NER, but also links the role of TFIIH in NER and transcription.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição TFII/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Viabilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFII/metabolismo , Raios UltravioletaRESUMO
Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.
Assuntos
Regulação da Expressão Gênica , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica , Enzimas de Conjugação de Ubiquitina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/química , Proteínas Inibidoras de STAT Ativados/genética , Multimerização Proteica , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/química , Lisina/química , Proteínas de Plantas/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciprofloxacina/toxicidade , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Microscopia de Força Atômica , Modelos Moleculares , Mutação , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestrutura , Multimerização Proteica , Solanum tuberosumRESUMO
The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.
Assuntos
Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição TFII/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Tolerância a Radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Proteína Supressora de Tumor p53/química , Raios UltravioletaRESUMO
DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reparo do DNA , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Ciprofloxacina , Quebras de DNA de Cadeia Dupla , DNA de Plantas/metabolismo , Rearranjo Gênico , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Plastídeos/genética , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
DNA double-strand breaks are highly detrimental genomic lesions that routinely arise in genomes. To protect the integrity of their genetic information, all organisms have evolved specialized DNA-repair mechanisms. Whirly proteins modulate DNA repair in plant chloroplasts and mitochondria by binding single-stranded DNA in a non-sequence-specific manner. Although most of the results showing the involvement of the Whirly proteins in DNA repair have been obtained in Arabidopsis thaliana, only the crystal structures of the potato Whirly proteins WHY1 and WHY2 have been reported to date. The present report of the crystal structures of the three Whirly proteins from A. thaliana (WHY1, WHY2 and WHY3) reveals that these structurally similar proteins assemble into tetramers. Furthermore, structural alignment with a potato WHY2-DNA complex reveals that the residues in these proteins are properly oriented to bind single-stranded DNA in a non-sequence-specific manner.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Plantas/química , Solanum tuberosum/química , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Cloroplastos/metabolismo , Sequência Conservada , Cristalografia por Raios X , DNA de Plantas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de SequênciaRESUMO
Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.
RESUMO
The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-ß-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-ß-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-ß-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8-10 carbons. Functional inhibition of the TGF-ß receptor (TGF-ßR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-ßR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-ßR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-ßR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-ßR1 in order to antagonize TGF-ß-mediated signaling that is required for ovarian cancer cell invasion and metastasis.
Assuntos
Catequina/farmacologia , Ácido Gálico/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Chá/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Catequina/química , Catequina/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Ácido Gálico/análogos & derivados , Ácido Gálico/isolamento & purificação , Humanos , Neoplasias Ovarianas/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismoRESUMO
Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.